Department of Chemistry, Ludwig-Maximilians University München, München, Germany.
Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians University München, Planegg-Martinsried, Germany.
Nat Methods. 2023 Apr;20(4):523-535. doi: 10.1038/s41592-023-01807-0. Epub 2023 Mar 27.
Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.
单分子Förster 共振能量转移（smFRET）实验允许在体外和体内研究生物分子的结构和动态。我们进行了一项涉及 19 个实验室的国际盲法研究，以评估 FRET 实验在测量 FRET 效率直方图、确定距离以及检测和量化结构动力学方面的不确定性。使用具有不同构象变化和动力学的两种蛋白质系统，我们获得了 FRET 效率的不确定性≤0.06，这对应于两个染料之间距离的精度≤2 Å 和准确度≤5 Å。我们进一步讨论了在这个距离范围内检测波动的限制以及如何识别染料扰动。我们的工作表明，smFRET 实验能够同时测量距离并避免对现实蛋白质系统构象动力学进行平均，突出了其在综合结构生物学不断扩展的工具包中的重要性。
